1. Field of the Invention
This invention relates to substrate analog inhibitors of tissue kallikrein which comprise peptide sequences corresponding to the partial amino acid sequence of kininogen. These substrate analogs specifically inhibit the activity of tissue kallikrein. Administration of these substrate analogs to a patient affects the biological processes associated with tissue kallikrein function including inflammation, edema, the regulation of blood flow, and the regulation of proenzyme activity through processing. In turn, this can control shock, hypotension and the perception of pain.
2. Description of the Background
Kallikreins are substrate specific proteolytic enzymes which are found in nearly xty years ago when Frey and Kraut observed that intravenous injections of human urine into dogs had hypotensive properties (E. K. Frey and H. Kraut, Arch. Exp. Pathol. Pharmakol. 133:1, 1926). These investigators determined that a specific substance was responsible. This substance was identified in human pancreatic cells and was termed "kallikrein," the greek synonym for pancreas ("kallikreas"; H. Kraut et al., Physiol. Chem. 189:97, 1930).
The kallikreins are serine proteases, a group of enzymes wherein each contains an active serine residue. About fifty serine proteases have been identified to date and there are likely to be many more (Bradykinin, Kallidin and Kallikrein, E. G. Erdos editor, Springer-Verlag, New York, N.Y., 1979).
There are two principle categories of kallikreins, the plasma kallikreins (Enzyme Commission of the International Union of Biochemistry Committee on Nomenclature or "EC" Number 3.4.21.34), and the tissue kallikreins (EC number 3.4.21.35), also referred to as glandular or organ kallikreins. In some animals, notably the rat and mouse, gene duplication has led to the appearance of many kallikreins. In higher animals, the number is more restricted. Plasma kallikrein, with a molecular weight of about 100,000 daltons, circulates in the blood in a precursor form called kallikreinogen or prekallikrein. Prekallikrein is converted to its activated form by factor XII of the blood clotting cascade, also called the Hageman Factor. The principle functions of plasma kallikrein are in the activation of the blood clotting and compliment enzyme cascades. In the clotting process, factor XII activates plasma kallikrein which, besides activating additional prekallikrein, activates intrinsic blood coagulation. Plasma kallikreins are also involved with the activation of the complement cascade which entails upwards of a dozen different proteases.
Tissue kallikreins have been isolated from saliva, intestine, lung, brain, plasma, and a variety of other bodily cells and fluids. The well-known types were isolated from urine and the pancreas. Biochemical studies reveal that tissue kallikreins are heat-stable glycoproteins which consist of a single amino acid chain having a molecular weight of 27,000 to 40,000 daltons. Both the amino acid sequence and the tertiary conformation of some kallikreins are known. Substrates of tissue kallikrein include procollagenase, kininogen, proinsulin, prorenin, BAM 22P, atrial naturietic factor, low density lipoprotein, atriopeptigen, and tissue plasminogen activator. These compounds are each processed or activated by proteolysis. Kininogen, in particular, requires kallikrein activity. Cleavage products of kininogens are referred to as kinins. Kinins are hormones which fall into the group called autocoids. Traditional hormones are made by a particular gland and act at sites distant from that gland. In contrast, autocoids are made locally, and bind and act locally.
Kinins liberated from kininogen bind to kinin receptors. It is generally believed that there are several types of kinin receptors. The major kinin binding structures that have been identified on normal cells are the B.sub.1, B.sub.2 (the classic kinin receptor), B.sub.3, B.sub.4 and B.sub.5 surface receptors. These receptors can also bind heterologous molecules (kinin analogs) found in bee and snake venom. A series of synthetic peptides based on the structure of bradykinin have been shown to block kinin receptors, and particularly the B.sub.2 receptor. These kinin analogs are not closely related to substrate analog inhibitors.
Numerous functions have been attributed to kinins including increased chloride transport across plasma membranes, the activation of phospholipase A.sub.2, and the release of interleukins, substance P, prostaglandins, and tumor necrosis factor. Primary roles for kinins have been shown to include the regulation of blood flow, blood pressure and sodium/water balance. Kinins have also been implicated in the sensation of pain, rheumatoid arthritis, allergic reactions, and vascular leakage (edema) from rhinitis. Clinical evidence indicates that administration of angiotensin I converting enzyme inhibitors (ACE), a known potentiator of kinin, produces a decrease in mean arterial pressure in humans. This correlate better with increases in circulating levels of kinins than with decreases in levels of circulating angiotensin II.
Tissue kallikreins are relatively specific for cleavage of kininogen, a hepatic-derived protein which circulates in the blood in two forms, a low molecular weight form of about 50,000 to 60,000 daltons (LMW-kininogen), and a high molecular weight form of about 120,000 daltons. Plasma kallikreins prefer to cleave the high molecular weight form, whereas tissue kallikreins prefer the low molecular weight form. Tissue kallikrein cleaves at two types of peptide bonds in LMW-kininogen, a methionine-lysine bond and an arginine-serine bond. Tissue kallikrein is thought to first cleave the arginine-serine peptide bond and then the methionine-lysine peptide bond to release Lys-bradykinin (Kallidine). Kallidine may be further processed by various amino peptidases into the nonapeptide, bradykinin.
Kallidin has been termed the "master hormone of inflammation" and one of the major roles of the tissue kallikreins in the body is in the regulation of inflammation. Inflammation is a multimediated process involving a large number of endogenous substances including the kinins. The kinins involved in inflammation include bradykinin, Lys-bradykinin or kallidin, and Met-Lys-bradykinin. They have the properties of being able to contract most smooth muscle preparations, but relax the muscles of the rat duodenum. Each of these peptides display similar actions, but with different potencies.
Trauma and local inflammation leads to an increased synthesis by the liver of certain plasma proteins, the acute phase reactants. Kininogen and kininogenase belong in this category of substances. It has been observed that total kininogen levels change in response to certain pathological conditions pointing to a possible involvement of kinin in the pathological process. However, local conditions can induce a continuous formation of kinins in the injured area. Therefore, it is more important to look at release of kinins in the extravascular tissues from extravasated plasma proteins in order to demonstrate the participation of these substances in inflammation.
Bradykinin, kallidin and related polypeptides are found in high concentrations at the sites of physiologically traumatic events. As trauma usually produces inflammation, a possible connection was investigated. Bradykinin applied to skin vessels of anaesthetized rats produced a marked increase in flow through the capillary vessels. Venules were dilated to a proportionately greater extent than were arterioles. Amounts less than one nanogram produced only vasomotor changes. Amounts of greater than one nanogram produced vasomotor changes were much more protracted and an increased permeability developed in the post capillaries and the collecting venules which was independent of histamine release. There was also a tendency for blood vessels to clump leading to a complete stasis of blood flow in these vessels.
Pain in the inflammatory process is a result of the release of algogenic or other substances which sensitize the pain receptors or P-fibers. Mechanical disruption is thought to play an important part in the development of pain, but pain receptors are not mechano-sensitive and pain and swelling are not related in a constant manner. Pain receptors are essentially chemoreceptive and found in the visceral and cutaneous areas of the body. Injury and regional ischaemia, including thermal injury, chemical injury, electrical injury, local trauma, and localized or systemic infection, leads to the disintegration of circulating and fixed cells. These cells release lysosomal proteases, produce localized acidosis, stimulate the production of prostaglandins and vasoactive amines, and activate the kinin system. All these conditions favor the stimulation and sensitization of pain receptors.
Involvement of kinins and kallikreins in inflammation produces vasodilation, increased vascular permeability, pain, and edema. Understanding the importance of tissue kallikrein in these biological processes depends on development of tissue kallikrein inhibitors which can be used in vivo. Specific inhibitors would reduce or decrease the undesirable effects of, for example, inflammation. Specific inhibition of tissue kallikrein would not inhibit the action of either the blood coagulation or complement cascades, either of which would be unwanted consequences of inhibition of plasma kallikrein.
In many instances, depletion of the available pool of kininogen reduces the signs and symptoms of inflammation. Kinin destroying enzymes, such as carboxypeptidase-B, has also been shown to interfere with the development of inflammation.
The kinin receptor blockers have been extremely valuable for the study of the biological effects of kinins. However, these compounds do not identify the source of kinins, making it difficult to know whether tissue or plasma kallikrein is responsible for a specific physiological effect. The receptor blockers also function as partial agonists in some systems, complicating the analysis of results from in vivo tests. Protease inhibitors such as aprotinin and its fragments, benzamidine, aromatic diamidines, and peptides containing a chloromethyl ketone group, do not appear to be specific for tissue kallikrein.
The key issue in the successful design of tissue kallikrein inhibitors is specificity. The progenitor serine protease which gave rise to tissue kallikrein also led to numerous other serine proteases. Tissue kallikrein and its family of related enzymes still share many features, including the ability to hydrolyze similar substrates and to be inhibited by related compounds such as aprotinin and the benzamidines. To avoid non-specific effects, tissue kallikrein inhibitors should not block other proteases.
A theory to explain specificity of proteases for their substrates has been developed (M. S. Deshpande and J. Burton, J. Med. Chem. 35:3094-3102, 1992). This theory points to a method of preparing specific inhibitors of a variety of proteases including tissue kallikrein. It is based on the observation that highly homologous serine proteases function specifically in vivo. Some factors makes it possible for proteases to selectively cleave substrates which are commingled in the blood such that each enzyme performs a single function.
The specificity of the interaction must reside in the match between the amino acid sequences of the cleavage site of the substrate and the active site of the enzyme. It has been shown that the species specificity of renin for angiotensinogen was mimicked by relatively short peptides from the cleavage site of the substrate (J. Burton and T. Quinn, Biochim. Biophys. Acta 952: 9-12, 1986). Subsequently, oligopeptides which encompass the cleavage site of kininogen were demonstrated to specifically bind to tissue kallikrein and much less well to related proteases (H. Okunishi et al., Hypertension 7:72-75, 1985).
The cleavage site can be depicted as a series of peptides numbered according to proximity from a central arginine residue. Beginning with arginine and continuing to the left, toward the amino terminus, residues are designated P.sub.1, P.sub.2, P.sub.3, etc. Residues to the right of arginine, toward the carboxy terminus, are designated P.sub.1 ', P.sub.2 ', P.sub.3 ', etc. (SEQ. ID. NO. 4):
______________________________________ 386 388 390 392 (amino acid position) Ser- Pro- Phe- Arg- Ser- Val- Gln- (bovine kininogen) P.sub.4 P.sub.3 P.sub.2 P.sub.1 .uparw. P.sub.1 ' P.sub.2 ' P.sub.3 ' ______________________________________
The premise that specificity resides in residues around the cleavage site (.uparw.) has been quantified by recent work with a series of kininogen peptides which bind to tissue kallikrein. The energy (.DELTA..DELTA.G) that each amino acid in the sequence (SEQ. ID. NO. 4) Ser-Pro-Phe-Arg-Ser-Val-Gln contributes to binding of tissue kallikrein is depicted in FIG. 1. Eighty percent of the energy of interaction comes from the Phe-Arg-Ser sequence at the core of the inhibitor (M. S. Deshpande et al., J. Med. Chem. 35:3094-3102, 1992). Tissue kallikrein is inhibited by peptides which are homologous with the amino acid sequence of the substrate around the cleavage site.
Tissue kallikrein and plasma kallikrein appear to recognize overlapping, but somewhat different parts of the kininogen molecule. Preliminary experiments on the interaction of the kininogen peptides with plasma kallikrein show that most of the binding energy for the kininogen-plasma kallikrein interaction may be attributed to the arginine (P.sub.1) and glutamine (P.sub.3 ') residues (G. Lalmanach et al., Proc. 13th Am. Pept. Symp., Edmonton, Alberta, Canada, June 1993). Neither the phenylalanine residue at P.sub.2 nor the serine residue at P.sub.1 ' appear to be important in the interaction between the substrate and this enzyme. Despite these distinctions, to date, no specific inhibitors of tissue kallikrein have been identified.